The ability to rapidly and efficiently generate a representative set of single copy sequences targeted to specific segments of the genome is important for genomic studies and cytogenetic analysis of chromosomal aberrations associated with genetic disorders, cancers and other diseases.
Traditional polymerase chain reaction (PCR) amplification methods have required knowledge of the sequences flanking both sides of the region that is to be amplified, so that the flanking sequences can serve as primers for the amplification procedure. Such a requirement has severely restricted use of this type of PCR as a method for generating amplification products from any region of the genome that is desired.
Using interspersed repetitive element (IRE) PCR procedures (referred to as inter-IRE PCR), in which both of the primers are derived from an IRE with known sequence, specific amplification of certain human genomic DNA sequences contained within somatic cell hybrids, yeast artificial chromosomes (YACs), cosmids and phage vectors have been reported. Inter-IRE PCR, however, is limited in that it will occur only between pairs of IRE sequences which are at an appropriate distance and orientation with respect to each other. The utility of inter-IRE PCR is further restricted by the apparent asymmetric distribution of repeat sequences within the genome. Such partitioning of repeats, resulting in regions which are relatively rich or poor with respect to their IRE content, often leads to a non-uniform distribution of IRE PCR products, leaving some regions of the genome significantly under-represented.
The use of a bubble primer in PCR has also been reported. Such use, however, has been limited to use in conjunction with a second primer which is derived from a unique region of the source DNA whose sequence and specific location relative to the region desired to be amplified are known. This method is thus also limited in that only regions of the genome in which the sequence of a specific adjacent region is known, can be amplified.